Camptothecin derivatives

ABSTRACT

A camptothecin derivative comprising a compound of the formula [I]:                    
     wherein R 1 , R 2 , R 3 , R 4  and R 5  are (A) adjacent two groups combine to form alkylene, or both are H, and one of the remaining three groups is —X n -Alk m -R 6 , and the other two are H, alkyl or halogen, or (B) adjacent two groups combine to form alkylene, and one of the carbon atoms of said alkylene group is substituted by —X n -Alk m -R 6 , and the remaining three groups are H, alkyl or a halogen, and one or two —CH 2 — of the alkylene in (A) or (B) may optionally be replaced by —O—, —S— or —NH—, X is —O— or —NH—, Alk is alkylene,                    
     or —OH, m and n are both 0 or 1, or m is 1 and n is 0, which camptothecin compound is bound to a polysaccharide having carboxyl groups via an amino acid or a peptide, or a pharmaceutically acceptable salt thereof. Said camptothecin derivatives show enhanced antitumor activities but few side effects and are useful as a medicament.

This application is a divisional a continuation-in-part of applicationSer. No. 09/227,158, filed on Jan. 8, 1999, U.S. Pat. No. 6,512,118which is a divisional of application Ser. No. 08/773,182 filed Dec. 27,1996, now U.S. Pat. No. 5,892,043, the entire contents of which arehereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a novel camptothecin derivative havingenhanced antitumor activities. More particularly, the present inventionrelates to a novel camptothecin derivative which is prepared bycombining an aminoalkoxy- or hydroxyalkoxy-camptothecin compound with apolysaccharide having carboxyl groups via an amino acid or a peptide,and a process for preparing the same. The camptothecin derivatives ofthe present invention can be delivered into a target region of thepatient selectively and in much amount, so that they can show desiredpharmacological activities at the desired region of the patient.Therefore, the antitumor activities of the camptothecin compounds areenormously enhanced and their side effects can be reduced, and hence,the camptothecin derivatives of the present invention are extremelyuseful as medicaments.

PRIOR ART

Camptothecin is one of several plant alkaloids, and has the followingformula:

and it has been known to show antileukemic and antitumor activities, andone camptothecin derivative, irinotecan hydrochloride {CPT-11,7-ethyl-10-[4-(piperidino)-1-piperidino]carbonyloxycamptothecin}, hasalready been put on the market. However, CPT-11 shows potent antitumoractivities in clinical use but also shows severe toxicity like otherantitumor agents, so that CPT-11 has been restricted in its therapeuticuse [cf. Cancer and Chemotherapy, vol. 21, p. 709 (1994)].

There have been synthesized various camptothecin compounds, and it hasbeen reported that these camptothecin compounds show antitumoractivities (Japanese Patent First Publication (Kokai) Nos. 279891/1989,222048/1993, 87746/1994, 228141/1994, and Japanese Patent FirstPublication (Kohyo) Nos. 503505/1992, 502017/1992).

On the other hand, in order to enhance antitumor activities and also toreduce the side effects thereof as low as possible, these compoundshaving such severe side effects have been studied as to a kind of drugdelivery system therefor, by which a necessary amount of a drug isselectively delivered into a target tissue. Especially, in thechemotherapy of cancers, it is a serious problem that there is nosignificant difference between tumor cells and normal cells insensitivity against anticancer agents, and many studies ontargeting-type drug delivery system for anticancer agents have been donein order to selectively deliver an anticancer agent into acancer-bearing region, for example, doxorubicin-polysaccharide complex(WO 94/19376), doxorubicin-inclusive liposome (Enhancement of effects ofanticancer agents and targeting therapy, p. 227 (1987), published byScience Forum Ltd.), dextran-binding mitomycin (Enhancement of effectsof anticancer agents and targeting therapy, p. 278 (1987), published byScience Forum Ltd.).

As explained above, camptothecin compounds show excellent anti-tumoractivities and are very useful as a medicament but they are strictlyrestricted in clinical use because of their severe side effects. Thus,it is desired to develop a new camptothecin derivative wherein theexcellent pharmacological activities of camptothecin compounds are dulyretained but undesirable severe side effects are suppressed.

Under the above mentioned circumstances, the present inventors haveintensively studied in order to obtain an excellent camptothecinderivative without the drawback of the conventional camptothecincompounds by utilizing the techniques of the above mentioned drugdelivery system, and finally have found that a novel camptothecinderivative having desired pharmacological effects can be obtained bycombining a camptothecin compound having a reactive group with apolysaccharide having carboxyl groups via an amino acid or a peptide,and have accomplished the present invention.

BRIEF DESCRIPTION OF INVENTION

An object of the present invention is to provide a novel camptothecinderivative comprising the camptothecin compound [I] bound to apolysaccharide having carboxyl groups via an amino acid or a peptide,which has enhanced antitumor activities with less side effects.

Another object of the present invention is to provide a process forpreparing these camptothecin derivative.

DETAILED DESCRIPTION OF INVENTION

The compound of the present invention is a camptothecin derivativecomprising a camptothecin compound having an aminoalkoxy group or ahydroxyalkoxy group, represented by the formula [I]:

wherein R¹, R², R³, R⁴ and R⁵ are

(A) among R¹, R², R³, R⁴ and R⁵, two groups being adjacent each othercombine to form an alkylene group, or both are a hydrogen atom, and oneof the remaining three groups of R¹, R², R³, R⁴ and R⁵ is a group of theformula: —X_(n)-Alk_(m)-R⁶, and the other two groups are a hydrogenatom, an alkyl group or a halogen atom, or

(B) among R¹, R², R³, R⁴ and R⁵, two groups being adjacent each othercombine to form an alkylene group, and one of the carbon atoms of saidalkylene group is substituted by a group of the formula:—X_(n)-Alk_(m)-R⁶, and the remaining three groups of R¹, R², R³, R⁴ andR⁵ are a hydrogen atom, an alkyl group or a halogen atom, and

one or two methylene groups of the alkylene group in (A) or (B) mayoptionally be replaced by —O—, —S— or —NH—,

X is —O— or —NH—,

Alk is an alkylene group,

R⁶ is —NH₂, a group of the formula:

m and n are both 0 or 1, or m is 1 and n is 0,

which camptothecin compound is bound to a polysaccharide having carboxylgroups via an amino acid or a peptide. The camptothecin derivative ofthe present invention shows extremely potent antitumor activities butshow low toxicity.

The camptothecin derivative of the present invention includes compoundswhich are prepared by combining the camptothecin compound [I] with apolysaccharide having carboxyl groups via an amino acid or a peptide.For example, such camptothecin derivatives may be prepared by combininga part or all of the carboxyl groups of an amino acid or a peptide withR⁶ of the compound [I] through acid-amide or ester bonds, followed bycombining a part or all of the carboxyl groups of a polysaccharide withamino groups of said amino acid or said peptide through acid-amidebonds. More particularly, the camptothecin derivative of the presentinvention includes compounds which are prepared by combining theC-terminal carboxyl group of an amino acid or a peptide with R⁶ of thecompound [I] through an acid-amide or ester bond, followed by combininga part or all of the carboxyl groups of a polysaccharide with theN-terminal amino group of said amino acid or said peptide throughacid-amide bonds.

Each substituent of the compound of the formula [I] of the presentinvention is explained below.

The alkylene group in the definition (A) formed by combining adjacenttwo groups of R¹, R², R³, R⁴ and R⁵ each other, wherein one or twomethylene groups may optionally be replaced by —O—, —S— or —NH—, isformed by combining each other two substituents at 7- and 9-positions,9- and 10-positions, 10- and 11-positions, or 11- and 12-positions ofthe formula [I], and the alkylene group includes a straight chain orbranched chain alkylene group having 2 to 6 carbon atoms, for example,ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene,methylmethylene, methylethylene, methyltrimethylene, etc.

The above alkylene group wherein one of the methylene groups is replacedby —O—, —S— or —NH— includes an alkylene group wherein a methylene groupat the end or at any position other than the end is replaced by —O—, —S—or —NH—. For example, such alkylene group includes an alkyleneoxy grouprepresented by the formula: —O-Alk′- (Alk′ is an alkylene group,hereinafter, the same) such as methyleneoxy, ethyleneoxy,trimethyleneoxy, tetramethyleneoxy, methylethyleneoxy; an alkyleneaminogroup represented by the formula: —NH-Alk′- such as methyleneamino,ethyleneamino, trimethyleneamino, tetramethyleneamino,methylethyleneamino; an alkylenethio group represented by the formula:—S-Alk′- such as methylenethio, ethylenethio, trimethylenethio,tetramethylenethio, methylethylenethio; an alkyleneoxyalkyl grouprepresented by the formula: -Alk′-O-Alk′- such as methyleneoxymethyl,ethyleneoxymethyl, trimethyleneoxymethyl, methylethyleneoxymethyl; analkyleneaminoalkyl group represented by the formula: -Alk′-NH-Alk′- suchas methyleneaminomethyl, ethyleneaminomethyl, trimethyieneaminomethyl,methylethyleneaminomethyl; an alkylenethioalkyl group represented by theformula: -Alk′-S-Alk′- such as methylenethiomethyl, ethylenethiomethyl,trimethylenethiomethyl, methylethylenethiomethyl, and the like.

The above alkylene group wherein two methylene groups are replaced by—O—, —S— or —NH— includes an alkylene group wherein two methylene groupsat the ends or at positions other than the ends are replaced by —O—, —S—or —NH—. For example, such alkylene group includes an alkylenedioxygroup represented by the formula: —O-Alk′-O— such as methylenedioxy,ethylenedioxy, trimethylenedioxy, tetramethylenedioxy,methylethylenedioxy; an alkylenediamino group represented by theformula: —NH-Alk′-NH— such as methylenediamino, ethylenediamino,trimethylenediamino, tetramethylenediamino, methylethylenediamino; analkylenedithio group represented by the formula: —S-Alk′-S— such asmethylenedithio, ethylenedithio, trimethylenedithio,tetramethylenedithio, methylethylenedithio, and the like.

The Alk in the group of the formula: —X_(n)-Alk_(m)-R⁶ includes astraight chain or branched chain alkylene group having 1 to 6 carbonatoms, for example, methylene, ethylene, trimethylene, tetramethylene,pentamethylene, hexamethylene, methylethylene, methyltrimethylene, etc.The group of the formula: —X_(n)-Alk_(m)-R⁶ is, for example, anaminoalkyloxy group (e.g. aminoethyloxy, aminopropyloxy), apiperazinylalkyloxy group (e.g. piperazinylethyloxy,piperazinylpropyloxy, piperazinylbutyloxy, piperazinylpentyloxy), ahydroxyalkyloxy group (e.g. hydroxyethyloxy, hydroxypropyloxy,hydroxybutyloxy, hydroxypentyloxy), an aminoalkylamino group (e.g.aminoethylamino, aminopropylamino, aminobutylamino, aminopentylamino), apiperazinylalkylamino group (e.g. piperazinylethylamino,piperazinylpropylamino, piperazinylbutylamino, piperazinylpentylamino),a hydroxyalkylamino group (e.g. hydroxyethylamino, hydroxypropylamino,hydroxybutylamino, hydroxypentylamino), an aminoalkyl group (e.g.aminomethyl, aminoethyl, aminopropyl, aminobutyl, aminopentyl), apiperazinylalkyl group (e.g. piperazinylmethyl, piperazinylethyl,piperazinylpropyl, piperazinylbutyl, piperazinylpentyl), a hydroxyalkylgroup (e.g. hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl,hydroxypentyl), amino group, piperazino group, and hydroxy group.

The alkylene group in the definition (B) formed by combining adjacenttwo groups of R¹, R², R³, R⁴ and R⁵ each other, wherein one or twomethylene groups may optionally be replaced by —O—, —S— or —NH—, and oneof the carbon atoms of said alkylene group is substituted by a group ofthe formula: —X_(n)-Alk_(m)-R⁶, is formed by combining each other twosubstituents at 7- and 9-positions, 9- and 10-positions, 10- and11-positions, or 11- and 12-positions of the formula [I]. Such alkylenegroup includes a straight chain or branched chain alkylene group having2 to 6 carbon atoms, for example, ethylene, trimethylene,tetramethylene, pentamethylene, hexamethylene, methylethylene,methyltrimethylene, etc., and the alkylene group wherein a methylenegroup or two methylene groups thereof are replaced by —O—, —S— or —NH—are the same ones as those exemplified in the above. Among thesealkylene groups, ones wherein one of the carbon atoms is substituted bya group of the formula: —X_(n)-Alk_(m)-R⁶ are also the same ones asthose exemplified in the above.

The lower alkyl group for the remaining groups of R¹, R², R³ ₁ R⁴ and R⁵which do not form an alkylene group includes a straight chain orbranched chain alkyl group having 1 to 6 carbon atoms, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,pentyl, hexyl, etc.

The halogen atom is fluorine, chlorine, bromine or iodine.

Moreover, examples of the partial structure formed by Ring A and Ring Bin the above definition (A) of the formula [I] are the followingstructures:

wherein X, Alk, R⁶, m and n are the same as defined above.

Besides, examples of the partial structure formed by Ring A and Ring Bin the above definition (B) of the formula [I] are the followingstructures:

wherein R¹, R², R³, R⁴ and R⁵ are each an alkyl group, a halogen atom ora hydrogen atom, and X, Alk, R⁶, m and n are the same as defined above.

Among them, the preferable combinations of the R¹, R², R³, R⁴ and R⁵are:

(1) R¹ and R² combine to form a trimethylene group, R³ is a3-amino-propyloxy group, R⁴ and R⁵ are each a hydrogen atom:

(2) R¹ is a piperazinomethyl group, R² and R⁵ are each a hydrogen atom,R³ and R⁴ combine to form an ethylenedioxy group:

(3) R¹ is an aminomethyl group, R² and R⁵ are each a hydrogen atom, R³and R⁴ combine to form an ethylenedioxy group:

(4) R¹, R², R⁴ and R⁵ are each a hydrogen atom, R³ is a 3-aminopropyloxygroup:

(5) R¹ and R² combine to form an amino-substituted trimethylene group,R³ is a methyl group, R⁴ is a fluorine atom, R⁵ is a hydrogen atom:

and

(6) R¹, R³, R⁴ and R⁵ are each a hydrogen atom, R² is an amino group:

The “polysaccharide having carboxyl groups” of the present inventionincludes the same as those as disclosed in the above mentioned WO94/19376, and includes polysaccharides originally having carboxyl groupsin the structure thereof (e.g. hyaluronic acid, pectic acid, alginicacid, chondroitin, heparin, etc.), and polysaccharides having originallyno carboxyl group (e.g. pullulan, dextran, mannan, chitin, mannoglucan,chitosan, etc.) but being introduced thereto carboxyl groups. Amongthese polysaccharides, dextran is especially preferable, particularlydextran having an average molecular weight of 20,000 to 400,000 is morepreferable, and particularly dextran having an average molecular weightof 50,000 to 150,000 is most preferable (said average molecular weightbeing determined by Gel permeation chromatography method (GPC analysis),Shinseikagaku Jikken Koza, vol. 20, p. 7). The polysaccharidesoriginally having no carboxyl group but being introduced theretocarboxyl groups mean ones which are prepared by replacing hydrogen atomsof a part or all of hydroxyl groups of polysaccharides originally havingno carboxyl groups by a carboxy-C₁₋₄ alkyl group.

The “polysaccharide having carboxyl groups” of the present inventionalso includes ones which are prepared by treating a polysaccharideoriginally having no carboxyl group with a reducing agent, and thenfollowed by replacing hydrogen atoms of a part or all of hydroxyl groupsof the product by a carboxy-C₁₋₄ alkyl group.

The alkyl moiety of the above carboxy-C₁₋₄ alkyl group which replacesthe hydrogen atoms of a part or all of hydroxyl groups of polysaccharidemay be either a straight chain alkyl group or a branched chain alkylgroup. Preferable carboxy-C₁₋₄ alkyl group is, for example,carboxymethyl, 1-carboxy-ethyl, 3-carboxypropyl,1-methyl-3-carboxypropyl, 2-methyl-3-carboxypropyl, 4-carboxybutyl,etc., and carboxymethyl and 1-carboxyethyl are more preferable.

In the present invention, the polysaccharide having carboxyl groups ispreferably a carboxymethylated dextran or pullulan.

When introducing a carboxyalkyl group into polysaccharides, the degreeof the introduction thereto is expressed by “degree of substitution”which is defined by the number of carboxyalkyl groups per a sugarresidue, i.e. expressed by the following equation.

Degree of Substitution=Number of carboxyalkyl groups in themolecule/Total number of sugar residues in the molecule

When the carboxyalkyl group is carboxymethyl group, the degree ofsubstitution is occasionally expressed by the degree ofcarboxymethylation (CM-degree).

When the polysaccharide is pullulan, dextran or mannoglucan, and all ofthe hydroxy groups thereof are substituted, the degree of substitutionthereof is 3, and the preferable degree of substitution is in the rangeof 0.3 to 0.8.

When the polysaccharide is chitin, and all of the hydroxyl groupsthereof are substituted, the degree of substitution thereof is 2, andthe preferable degree of substitution is in the range of 0.3 to 0.8.

Besides, it is essential that the polysaccharide of the presentinvention should have at least one carboxyalkyl group in the moleculeexcept for polysaccharides having originally carboxyl groups. Therefore,polysaccharides having a degree of substitution of 0 should be excludedfrom the polysaccharide of the present invention.

The polysaccharide having carboxyl groups may be prepared by the methoddisclosed in WO 94/19376.

The amino acid which intervenes between a camptothecin compound [I] anda polysaccharide having carboxyl groups includes both natural aminoacids and synthetic amino acids (including D-amino acid, L-amino acid, amixture thereof), and also includes either neutral amino acids, basicamino acids or acidic amino acids. Moreover, the amino acid of thepresent invention may be not only α-amino acids but also β-amino acids,γ-amino acids, ε-amino acids, etc., and includes, for example, glycine,α-alanine, β-alanine, valine, leucine, isoleucine, serine, threonine,cysteine, methionine, aspartic acid, glutamic acid, lysine, arginine,phenylalanine, tyrosine, histidine, tryptophan, proline, hydroxyproline,γ-aminobutyric acid, ε-amino-caproic acid, etc.

The peptide of the present invention includes peptides derived from theabove amino acids, or peptides having compounds other than amino acidsin the part of the chain thereof. For example, a dicarboxylic acid suchas succinic acid, a diamine such as ethylenediamine, or a diol such asethyleneglycol may exist in the middle of the peptide chain or theterminus of the peptide chain. Besides, the binding site of the peptidechain to the carboxyl groups of the polysaccharide usually starts fromthe N-terminus of the peptide chain through acid-amide bonds. When abasic amino acid (e.g. lysin) exists in the peptide chain, the bindingsite of the peptide chain may be reversed by binding an ε-amino group ofthe basic amino acid with carboxyl groups of a polysaccharide, andbinding an α-amino group with the C-terminus of the peptide chain.

Such peptides may be ones composed of two or more amino acids, i.e. oneshaving two or more peptide chains, more preferably ones having 2 to 5peptide chains. Suitable examples of the peptide chain are -Gly-Gly-L-or D-Phe-Gly- (SEQ ID NOS: 3 or 4, respectively), -Gly-Gly-,-Gly-Gly-Gly-, -Gly-Gly-Gly-Gly- (SEQ ID NO: 1), -Gly-Gly-Gly-Gly-Gly-(SEQ ID NO: 2), -L- or D-Phe-Gly-, -L- or D-Tyr-Gly-, -L- or D-Leu-Gly-,and peptide chains containing these sequences (the N-terminus of thesepeptides or peptide chains containing these sequences is introduced ontocarboxyl groups of a polysaccharide). Among these peptides, -Gly-Gly-L-or D-Phe-Gly-, -Gly-Gly-, -Gly-Gly-Gly-, -Gly-Gly-Gly-Gly- (SEQ ID NO:1), -Gly-Gly-Gly-Gly-Gly- (SEQ ID NO: 2), -L- or D-Phe-Gly- and -L- orD-Leu-Gly- are more preferable. Most preferable peptides are-Gly-Gly-L-Phe-Gly- (SEQ ID NO: 3), -Gly-Gly-, -Gly-Gly-Gly-,-Gly-Gly-Gly-Gly- (SEQ ID NO: 1) and -L- or D-Phe-Gly-.

The camptothecin derivatives of the present invention may usually beprepared by combining the compound [I] with an amino acid or a peptide,followed by reacting the product with a polysaccharide having carboxylgroups.

In the reaction between the compound [I] and an amino acid or a peptide,when R⁶ of the formula [I] is —NH₂ or a piperazino group, the compound[I] is combined with the C-terminal carboxyl group of an amino acid or apeptide through acid-amide bonds. When R⁶ of the formula [I] is —OH, thecompound [I] is combined with the C-terminal carboxyl group of an aminoacid or a peptide through ester bonds. In this case, it is preferable toprotect other functional groups of an amino acid or a peptide which donot participate in said acid-amide bonds or ester bonds, for example,the N-terminal amino group or other carboxyl groups, are protected in aconventional manner, prior to the reaction of the compound [I] and anamino acid or a peptide. The protecting group may be any protectinggroups which are conventionally used for protection of amino acids, andthe protecting group of amino group is, for example, t-butoxycarbonylgroup, p-methoxybenzyloxycarbonyl group, etc., and the protecting groupof carboxyl group is, for example, a lower alkyl group (e.g. t-butylgroup), benzyl group, etc.

The production of the above mentioned acid-amide bonds or ester bondsbetween R⁶ of the compound [I] and an amino acid or a peptide is carriedout by a conventional method, for example, by reacting in the presenceof a condensing agent in a suitable solvent. The solvent includes, forexample, dimethylformamide, tetrahydrofuran, etc., and the condensingagent includes, for example, dicyclohexylcarbodiimide,1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, etc.

The camptothecin compound thus prepared by combining the compound [I]with an amino acid or a peptide, after removing the protecting group ofan amino group or a carboxyl group therefrom by a conventional methodwhen an amino group or a carboxyl group thereof is protected, is reactedwith a polysaccharide having carboxyl groups, to give the desiredcamptothecin derivatives of the present invention. In this reaction, apart or all of the carboxyl groups of the polysaccharide are combinedwith the N-terminal amino group of the amino acid or the peptide whichis previously bonded to the camptothecin compound [I], throughacid-amide bonds.

The reaction of the camptothecin compound which is produced by combiningthe compound [I] with an amino acid or a peptide, and a polysaccharidehaving carboxyl groups is carried out by a conventional method, forexample, in the presence of a condensing agent in a suitable solvent.The solvent includes, for example, water, ethanol, dimethylformamide, ora mixture thereof, and the condensing agent includes, for example,1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride,2-ethyloxy-1-ethyloxycarbonyl-1,2-dihydroquinoline, etc.

In the camptothecin derivatives of the present invention, the ratio ofthe polysaccharide and the camptothecin compound [I] which is an activeingredient may be selected according to the kinds of the polysaccharideto be used, but the content of the camptothecin compound [I] in thecamptothecin derivative of the present invention is preferably in thefollowing range.

When the polysaccharide is pullulan, dextran, chitin, mannoglucan orN-acetyl-de-N-sulfuric heparin, it is in the range of 0.1 to 20% byweight, more preferably in the range of 2 to 10% by weight.

When dextran is used as a polysaccharide in the present invention, theaverage molecular weight of the camptothecin derivative of the presentinvention is preferably in the range of 30,000 to 500,000, morepreferably, in the range of 60,000 to 200,000, determined by the GPCanalysis.

The camptothecin derivatives of the present invention thus obtained maybe converted into a pharmaceutically acceptable salt thereof, ifnecessary. The pharmaceutically acceptable salt includes, for example,salts with an alkali metal or an alkaline earth metal (e.g. sodium salt,potassium salt, calcium salt, etc.), or salts with an amino acid (e.g.arginine salt, lysine salt, etc.).

Some of the camptothecin compounds of the formula [I] of the presentinvention are known compounds, for example, compounds disclosed inJapanese Patent First Publication (Kokai) Nos. 279891/1989, 222048/1993,87746/1994, 228141/1994 and Japanese Patent First Publication (Kohyo)Nos. 503505/1992, 502017/1993, etc., and may be prepared by conventionalmethods, such as by the method disclosed in the following ReactionScheme 1:

wherein R¹, R², R³, R⁴ and R⁵ are the same as defined above, R^(1′),R^(2′), R^(3′), R^(4′), and R^(5′) are the same as R¹, R², R³, R4 andR⁵, except that an amino group, a piperazino group or a hydroxyl groupin the group of the formula: —X_(n)-Alk_(m)-R⁶, which is contained inone of R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) is protected.

That is, the aminocarbonyl compound (1) is condensed with a knownpyranoindolidine (2) (cf. EP-0220601-A) by a method known as Friedländercondensation reaction (cf. Organic Reactions, 28, pp. 37-202, John Wiley& Sons, Inc., New York (1982)), followed by removing the protectinggroups from the product to give the camptothecin compound [I].

In the above process, a group of the formula: —X_(n)-Alk_(m)-R⁶, whichis contained in one of R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) may beintroduced after said Friedländer condensation reaction, when m is 1,and n is 1.

That is, in the process of the above Reaction Scheme 1, a compound ofthe formula (1) wherein a corresponding group to a group of the formula:—X_(n)-Alk_(m)-R⁶ is a hydroxyl group (—OH) or an amino group (—NH₂) isused, and condensed with the compound (2) by Friedländer condensationreaction, and the resulting condensed product is reacted with aprotected aminoalkanol or hydroxyalkanol represented by the formula:R^(6′)-Alk_(m)—OH (R^(6′) is a protected amino group, a protectedpiperazino group, or a protected hydroxy group, and Alk and m are thesame as defined above) or a reactive derivative thereof (e.g. aprotected aminoalkyl halide, a protected hydroxyalkyl halide), followedby removing the protecting groups therefrom to give the desiredcamptothecin compound [I].

The starting aminocarbonyl compound (1) may be prepared by the followingReaction Scheme 2:

wherein R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) are the same asdefined above.

The hydroxyl compound (a) is treated with an oxidizing agent such aspyridinium dichromate to give a ketone compound (b), which is furthersubjected to catalytic reduction in the presence of a suitable catalystsuch as palladium-carbon to give the compound (1).

When both m and n are 1, the group of the formula: —X_(n)-Alk_(m)-R⁶,which is contained in one of R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′),may be introduced into the compound (a) by reacting a compound of theformula (a) wherein a corresponding group to a group of the formula:—X_(n)-Alk_(m)-R⁶ is a hydroxyl group (—OH) or an amino group (—NH₂)with a compound of the formula: HO-Alk-R⁶ (Alk and R⁶ are the same asdefined above) or a reactive derivative thereof (e.g. a substitutedalkyl halide).

The camptothecin derivatives of the present invention and apharmaceutically acceptable salt thereof show excellent antitumoractivities against various tumors, especially they show excellenttherapeutic effects on solid tumors such as pulmonary cancer, uterinecancer, ovarian cancer, breast cancer, gastrointestinal cancer (largebowel cancer, gastric cancer, etc.).

The camptothecin derivatives of the present invention and apharmaceutically acceptable salt thereof are preferably administeredparenterally (e.g. intravascular injection), and are usually used in theform of a liquid preparation (e.g. solution, suspension, emulsion,etc.).

The dosage of the camptothecin derivatives of the present inventionvaries according to the administration method, ages, weights orconditions of the patients, but it is usually in the range of 0.02-50mg/kg/day, more preferably in the range of 0.1-10 mg/kg/day, convertedinto the dose of the camptothecin compound [1] (when R⁶ is —NH₂, ahydrochloride of the camptothecin compound [I], and when R⁶ is a groupof the formula:

a hydrochloride or dihydrochloride of the camptothecin compound [I]).

The camptothecin derivatives of the present invention and a process forpreparing thereof are illustrated in more detail by the followingExamples, but should not be construed to be limited thereto.

EXAMPLE 1 Preparation of the Camptothecin Derivative of the FollowingFormula (Where -Gly-Gly-L-Phe-Gly- is SEQ ID NO: 3)

CM-Dextran Na: Carboxymethyldextran Sodium Salt

(1) Preparation of 3-(t-Butoxycarbonylamino)propanol

3-Aminopropanol (6.0 g) is dissolved in methylene chloride (50 ml), andthereto is added dropwise with stirring di-t-butyl dicarbonate (18.3 g)under ice-cooling. The mixture is stirred at room temperature for 12hours, and concentrated, and the residue is purified by silica gelcolumn chromatography to give the title compound (13.98 g) as acolorless oil.

Yield: 99.9%; IR (Neat): ν_(max) ^(cm−1)=3380, 1790; Mass: m/z=176([M+H]⁺); NMR (300 MHz, CDCl₃): δ^(TMS)=1.45 (9H, s), 1.62-1.72 (2H, m),3.0 (1H, brs), 3.29 (2H, dd, J=12 Hz, 6 Hz), 3.66 (2H, dd, J=12 Hz, 6Hz), 4.80 (1H, brs).

(2) Preparation of 3-(t-Butoxycarbonylamino)propyl Tosylate

3-(t-Butoxycarbonylamino)propanol (10.0 g) is dissolved in methylenechloride (100 ml), and thereto are added with stirring triethylamine(8.66 g) and tosyl chloride (16.3 g) under ice-cooling, and the reactionmixture is stirred at room temperature overnight. The reaction mixtureis concentrated, and the residue is dissolved in a mixture of water andethyl acetate. The organic layer is separated, washed with a saturatedsodium chloride solution, dried over sodium sulfate, and concentratedunder reduced pressure. The residue is purified by silica gel columnchromatography to give the title compound (15.37 g) as a pale yellowoil.

Yield: 82%; IR (Neat): ν_(max) ^(cm−1)=3400, 3340, 1700; Mass: m/z=352([M+Na]⁺); NMR (300 MHz, CDCl₃): δ^(TMS)=1.42 (9H, s), 1.78-1.90 (2H,m), 2.45 (3H, s), 3.11-3.22 (2H, m), 4.09 (2H, t, J=6 Hz), 4.5-4.65 (1H,m), 7.36 (2H, d, J=8 Hz), 7.77-7.83 (2H, m).

(3) Preparation of5-[3′-(t-Butoxycarbonylamino)propyloxy]-1-hydroxy-8-nitro-1,2,3,4-tetrahydronaphthalene

1,5-Dihydroxy-8-nitro-1,2,3,4-tetrahydronaphthalene (2.0 g) (J. Med.Chem., 1973, 16 (3), 254) is dissolved in dry DMF (80 ml), and theretoare added potassium carbonate (2 equivalents ), sodium iodide (1.4equivalent) and 3-(t-butoxycarbonylamino)propyl tosylate (1.4equivalent). The reaction mixture is stirred at 50° C. for 24 hours, andthereto is added ethyl acetate. The mixture is washed with a saturatedaqueous sodium chloride solution, and dried over sodium sulfate. Theresidue is purified by silica gel column chromatography to give thetitle compound (3.05 g) as a pale yellow amorphous powder.

Yield: 87%; IR (Neat): ν_(max) ^(cm−1)=3360, 1695; Mass: m/z=384([M+NH₄]⁺); NMR (300 MHz, CDCl₃): δ^(TMS)=1.36 (9H, s), 1.57-1.90 (6H,m), 2.52-2.71 (2H, m), 3.11 (2H, q, J=6 Hz), 4.07 (2H, t,=6 Hz),5.12-5.17 (2H, m), 6.89 (1H, t, J=5.5 Hz), 6.96 (1H, d, J=9 Hz), 7.68(1H, d, J=9 Hz).

(4) Preparation of5-[3′-(t-Butoxycarbonylamino)propyloxy]-8-nitro-1,2,3,4-tetrahydronaphthalen-1-one

5-[3′-(t-Butoxycarbonylamino)propyloxy]-1-hydroxy-8-nitro-1,2,3,4-tetrahydronaphthalene(2.46 g) is dissolved in dry methylene chloride (110 ml), and theretoare added molecular sieves 3A (6.73 g) and pyridinium dichlorochromate(1.5 equivalent), and the mixture is refluxed. After the reaction iscompleted, the mixture is diluted with ether, and the insolublematerials are removed by filtration through a pad of Celite. Thefiltrate is concentrated, and the residue is purified by silica gelcolumn chromatography to give the title compound (1.87 g) as a colorlesspowder.

M.p. 76-77° C.; Yield: 76%; IR (Melt): ν_(max) ^(cm−1)=3550, 1700; Mass:m/z=382 ([M+NH₄]⁺); NMR (300 MHz, CDCl₃): δ^(TMS)=1.44 (9H, s),2.02-2.20 (4H, m), 2.68-2.73 (2H, m), 2.92 (2H, t, J=6 Hz), 3.36 (2H, q,J=6.5 Hz), 4.12 (2H, t, J=6 Hz), 4.78 (1H, brs), 6.95 (1H, d, J=9 Hz),7.39 (1H, d, J=9 Hz).

(5) Preparation of8-Amino-5-[3′-(t-butoxycarbonylamino)propyloxy]-1,2,3,4-tetrahydronaphthalen-1-one

5-[3′-(t-Butoxycarbonylamino)propyloxy]-8-nitro-1,2,3,4-tetrahydronaphthalen-1-one(3.55 g) is dissolved in ethanol (160 ml), and thereto is added 10%palladium-carbon (420 mg). The mixture is stirred under hydrogenatmosphere for 1.5 hour, and the catalyst is removed by filtration. Thefiltrate is concentrated, and the residue is purified by silica gelcolumn chromatography to give the title compound (3.56 g) as a yellowoil.

M.p.: 112-115° C.; Yield: 83%; IR (Nujol): ν_(max) ^(cm−1)=3440, 3340,1700, 1650; Mass: m/z=335 ([M+H]⁺); NMR (300 MHz, CDCl₃): δ^(TMS)=1.45(9H, s), 1.92-2.67 (4H, m), 2.61 (2H, t, J=6 Hz), 2.87 (2H, t, J=6 Hz),3.35 (2H, q, J=6.5 Hz), 3.94 (2H, t, J=6 Hz), 4.85 (1H, brs), 6.10 (2H,brs), 6.48 (1H, d, J=9 Hz), 6.94 (1H, d, J=9 Hz).

(6) Preparation of10-[3′-(t-Butoxycarbonylamino)propyloxy]-7,9-trimethylene-(20S)-camptothecin

8-Amino-5-[3′-(t-butoxycarbonylamino)propyloxy]-1,2,3,4-tetrahydronaphthalen-1-one(2.03 g) is dissolved in ethanol (85 ml), and thereto are added(4S)-7,8-dihydro-4-ethyl-4-hydroxy-1H-pyrano[3,4-f]indolidine-3,6,10(4H)-trione(800 mg) and p-toluenesulfonic acid (58 mg), and the mixture is refluxedfor 17 hours. After the reaction is completed, the mixture isconcentrated under reduced pressure to remove the solvent, and theresulting residue is purified by silica gel column chromatography togive the title compound (850 mg) as a pale yellow powder.

M.p.: 225-227° C. (decomposed); Yield: 50%; IR (Nujol): ν_(max)^(cm−1)=3440, 3325, 1750, 1740, 1655, 1620; Mass: m/z=562 ([M+H]⁺); NMR(300 MHz, CDCl₃): δ^(TMS)=1.03 (3H, t, J=7.5 Hz), 1.45 (9H, s),1.82-2.18 (6H, m), 3.06-3.13 (4H, m), 3.41 (2H, q, J=6 Hz), 3.79 (1H,s), 4.24 (2H, t, J=6 Hz), 4.9 (1H, br), 5.16 (2H, s), 5.30 (1H, d, J=16Hz), 5.75 (1H, d, J=16 Hz), 7.51 (1H, d, J=9 Hz), 7.61 (1H, s), 8.06(1H, d, J=9 Hz).

(7) Preparation of10-(3′-Aminopropyloxy)-7,9-trimethylene-(20S)-camptothecin Hydrochloride

10-[3′-(t-Butoxycarbonylamino)propyloxy]-7,9-trimethylene-(20S)-camptothecin(836 mg) is suspended in dioxane (30 ml), and thereto is added dropwisewith stirring a 18% hydrochloric acid in dioxane (15 ml) underice-cooling. The reaction mixture is stirred at room temperature, andafter the reaction is completed, isopropyl ether is added to thereaction mixture, and stirred. The precipitated powder is collected byfiltration, washed with ether, and dried under reduced pressure. Theyellow powder thus obtained is dissolved in water, and lyophilized togive the title compound (620 mg) as a yellow powder.

M.p.: >194° C. (decomposed); Yield: 84%; IR (Nujol): ν_(max)^(cm−1)=1740, 1655; Mass: m/z=462 ([M−Cl]⁺); NMR (300 MHz, d₆-DMSO):δ^(TMS)=0.88 (3H, t, J=7.5 Hz), 1.81-1.94 (2H, m), 1.97-2.15 (4H, m),3.01-3.14 (6H, m), 4.28 (2H, t, J=6 Hz), 5.23 (2H, s), 5.43 (2H, s),7.28 (1H, s), 7.71 (1H, d, J=9.5 Hz), 7.95-8.08 (3H, brs), 8.03 (1H, d,J=9.5 Hz).

(8) Preparation of10-[3′-(t-Butoxycarbonylglycylglycyl-L-phenylalanyl-glycylamino)propyloxy]-7,9-trimethylene-(20S)-camptothecin

10-(3′-Aminopropyloxy)-7,9-trimethylene-(20S)-camptothecin hydrochloride(158 mg) and diisopropylethylamine (49 mg) are dissolved with stirringin DMF (5 ml), and thereto is added a solution oft-butoxycarbonyl-glycylglycyl-L-phenylalanylglycine (278 mg) andN-hydroxysuccinimide (143 mg) in dry DMF (8 ml), and further addedthereto with stirring 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimidehydrochloride (183 mg) under ice-cooling. The mixture is stirred at roomtemperature for 16 hours. After the reaction is completed, the mixtureis concentrated under reduced pressure to remove the solvent, and theresulting residue is purified by silica gel column chromatography togive the title compound (285 mg) as a pale yellow powder.

Yield: Quantitative; IR (Nujol): ν_(max) ^(cm−1)=3290, 1660; Mass: m/z880 ([M+H]⁺); NMR (300 MHz, CDCl₃): δ^(TMS)=1.02 (3H, t, J=7.5 Hz), 1.43(9H, s), 1.85-1.94 (2H, m), 2.02-2.10 (4H, m), 2.97-3.05 (5H, m), 3.23(1H, dd, J=14 Hz, 5 Hz), 3.49 (2H, q, J=6.5 Hz), 3.60-3.80 (6H, m), 4.20(1H, t, J=6 Hz), 4.50-4.56 (1H, m), 5.11 (2H, s), 5.29 (1H, d, J=16.5Hz), 5.71 (1H, d, J=16.5 Hz), 5.85 (1H, brt), 7.08 (1H, m), 7.18-7.27(5H, m), 7.45 (1H, d, J=7 Hz), 7.52 (1H, d, J=9.5 Hz), 7.58 (1H, s),7.71 (1H, m), 7.99 (1H, d, J=9.5 Hz).

(9) Preparation of10-[3′-(Glycylglycyl-L-phenylalanylglycylamino)propyl-oxy]-7,9-trimethylene-(20S)-camptothecinhydrochloride

10-[3′-(t-Butoxycarbonylglycylglycyl-L-phenylalanylglycylamino)-propyloxy]-7,9-trimethylene-(20S)-camptothecin(273 mg) is dissolved in dioxane (10 ml), and thereto is added dropwisewith stirring 18% hydrochloric acid in dioxane (15 ml) underice-cooling. The reaction mixture is stirred at room temperature, andafter the reaction is completed, to the mixture is added isopropylether. The mixture is stirred, and the precipitated powder is collectedby filtration, washed with ether, and dried under reduced pressure. Theyellow powder thus obtained is dissolved in water, and lyophilized togive the title compound (210 mg) as a yellow powder.

M.p.: >174° C. (decomposed); Yield: 83%; IR (Nujol): ν_(max)^(cm−1)=3190, 1745, 1650; Mass: m/z=780 ([M−Cl]⁺); NMR (300 MHz,d₆-DMSO): δ^(TMS)=0.89 (3H, t, J=7 Hz), 1.26-1.32 (2H, m), 1.86-2.04(6H, m), 2.79 (1H, dd, J=14 Hz, 10 Hz), 2.98-3.05 (5H, m), 3.28-3.36(2H, m), 3.54-3.88 (6H, m), 4.20 (2H, t, J=6 Hz), 4.45-4.54 (1H, m),5.19 (2H, s), 5.43 (2H, s), 7.11-7.27 (5H, m), 7.35 (1H, s), 7.71 (1H,t, J=9.5 Hz), 7.97 (1H, t, J=5.5 Hz), 8.03 (1H, d, J=9.5 Hz), 8.19 (3H,br), 8.35 (1H, t, J=6 Hz), 8.43 (1H, d, J=8 Hz), 8.65 (1H, t, J=5.5 Hz).

The camptothecin compound having an amino group obtained by the aboveprocess is condensed with a water soluble high molecular compound havingcarboxyl groups such as carboxymethyldextran (hereinafter, referred toas CM-dextran) to give the desired camptothecin derivative. Thecondensation reaction is carried out in the presence of a condensingagent such as a water soluble carbodiimide[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, etc.] in a water or amixture of water-an organic solvent.

(10) Preparation of the Camptothecin Derivative

CM-Dextran sodium salt (CM-degree; 0.5) (500 mg) is dissolved in water(20 ml), and thereto is added with stirring10-[3′-(glycyl-glycyl-L-phenylalanyl-glycylamino)propyloxy]-7,9-trimethylene-(20S)-camptothecinhydrochloride (50 mg) at a temperature below 10° C. To the mixture isadded 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5g), during which the pH value of the mixture is kept at pH 6.5-7.0 with0.1 N hydrochloric acid. The mixture is stirred at a temperature below10° C. for two hours, and the pH value thereof is adjusted to pH 9 with0.1 N sodium hydroxide. The reaction mixture is purified by ion-exchangecolumn chromatography (AGMP-50, Na-type, 30 ml, manufactured by Bio-Rad,Laboratories, Inc.). The fractions containing the desired compound arecombined (30 ml), and thereto is added a 3M aqueous sodium chloridesolution (1.2 ml), and the mixture is poured into ethanol (150 ml). Theprecipitates are collected by centrifugation, and water (20 ml) is addedto the precipitate, and then the mixture is filtered. To the filtrate isadded a 3M aqueous sodium chloride solution (0.4 ml), and the mixture isadded with stirring to ethanol (80 ml). The precipitates are collectedby centrifugation, washed with the solvent, and dried under reducedpressure to give the desired camptothecin derivative (415 mg). Thecontent of 10-(3′-aminopropyloxy)-7,9-trimethylene-(20S)-camptothecinhydrochloride (the compound of Example 1-(7)) in the desiredcamptothecin derivative is 4.4% which is calculated on the basis of theabsorbance at 380 nm. According to the analysis by gel permeationchromatography (GPC), the average molecular weight of the desiredcamptothecin derivative is 160,000, and the degree of distribution(Mw/Mn) is 1.57.

Conditions for GPC analysis: G4000SWXL (manufactured by Toso, Ltd), 0.2Mphosphate buffer (pH 7.0).

EXAMPLE 2 Preparation of the Camptothecin Derivative of the FollowingFormula (Where -Gly-Gly-L-Phe-Gly- is SEQ ID NO: 3):

(1) Preparation of7-(4′-(t-Butoxycarbonylglycylglyclyl-L-phenylalanyl-glycyl)piperazino)methyl-10,11-ethylenedioxy-(20S)-camptothecin

The title compound (518 mg) is obtained in the same manner as in Example1-(8) as a yellow powder from7-piperazinomethyl-10,11-ethylene-dioxy-(20S)-camptothecin hydrochloride(450 mg) and t-butoxycarbonyl-glycyl-glycyl-L-phenylalanylglycine (2equivalents).

Yield: 74%; IR (Nujol): ν_(max) ^(cm−1)=3280, 1750, 1655; Mass: m/z=923([M+H]⁺); NMR (300 MHz, d₆-DMSO): δ^(TMS)=0.89 (3H, t, J=7.5 Hz), 1.37(9H, s), 1.85-2.1 (2H, m), 2.3-2.6 (4H, m), 2.75 (1H, dd, J=14 Hz, 10Hz), 3.05 (1H, dd, J=14 Hz, 4.5 Hz), 3.3-3.6 (6H, m), 3.58 (1H, dd, J=21Hz, 5.5 Hz), 3.74 (1H, dd, J=17 Hz, 5.5 Hz), 3.9-4.1 (4H, m), 4.44 (4H,s), 4.58 (1H, m), 5.24 (2H, s), 5.42 (2H, s), 6.50 (1H, s), 6.97 (1H, t,J=6 Hz), 7.1-7.3 (6H, m), 7.55 (1H, s), 7.77 (1H, s), 7.8-7.9 (1H, br),8.05-8.2 (2H, m).

(2) Preparation of7-(4′-(Glycylglycyl-L-phenylalanylglycyl)piperazino)-methyl-10,11-ethylenedioxy-(20S)-camptothecinHydrochloride

The title compound (409 mg) is obtained in the same manner as in Example1-(9) as a yellow powder from7-(4′-(t-butoxycarbonylgtycylglycyl-L-phenylalanylglycyl)piperazino)methyl-10,11-ethylenedioxy-(20S)-camptothecin(478 mg).

M.p. 237-239° C. (decomposed); IR (Nujol): ν_(max) ^(cm−1)=3250, 1745,1655; Mass: m/z=823 ([M−Cl]+); NMR (300 MHz, d₆-DMSO): δ^(TMS)=0.88 (3H,t, J=7 Hz), 1.8-1.99 (2H, m), 2.79 (1H, dd, J=14 Hz, 10 Hz), 3.07 (1H,dd, J=14 Hz, 4 Hz), 3.1-4.3 (16H, m), 4.47 (4H, s), 4.55-4.70 (1H, m),5.44 (2H, s), 5.67 (2H, s), 7.15-7.32 (6H, m), 7.65 (1H, s), 8.05 (1H,s), 8.05-8.20 (3H, br), 8.29 (1H, br), 8.39 (1H, d, J=8.5 Hz), 8.57 (1H,t, J=5.5 Hz).

(3) Preparation of the Camptothecin Derivative

CM-Dextran sodium salt (CM-degree; 0.5) (1.2 g) and7-(4′-(glycyl-glycyl-L-phenylalanylgycyl)piperazino)methyl-10,11-ethylenedioxy-(20S)-camptothecinhydrochloride (168 mg) are treated in the same manner as in Example1-(10) to give the desired camptothecin derivative (798 mg) as a paleyellow powder. The content of7-piperazinomethyl-10,11-ethylenedioxy-(20S)-camptothecin hydrochloridein the desired camptothecin derivative is 1.1% which is calculated onthe basis of the absorbance at 380 nm. According to the analysis by gelpermeation chromatography (GPC), the average molecular weight of thedesired camptothecin derivative is 169,000, and the degree ofdistribution (Mw/Mn) is 1.32.

Conditions for GPC analysis: G4000SWXL (manufactured by Toso, Ltd), 0.2Mphosphate buffer (pH 7.0).

EXAMPLE 3 Preparation of the Camptothecin Derivative of the FollowingFormula (Where -Gly-Gly-L-Phe-Gly- is SEQ ID NO: 3):

(1) Preparation of7-N-(t-Butoxycarbonylglycylglyclyl-L-phenylalanyl-glycyl)aminomethyl-10,11-ethylenedioxy-(20S)-camptothecin

The title compound (232 mg) is obtained in the same manner as in Example1-(8) as a yellow powder from7-aminomethyl-10,11-ethylenedioxy-(20S)-camptothecin hydrochloride (222mg) and t-butoxycarbonyl-glycyl-glycyl-L-phenylalanylglycine (2equivalents).

Yield: 58%; IR (Nujol): ν_(max) ^(cm−1)=3285, 1750, 1650; Mass: m/z=854([M+H]⁺); NMR (300 MHz, d₆-DMSO): δ^(TMS)=0.88 (3H, t, J=7.5 Hz), 1.35(9H, s), 1.78-1.94 (2H, m), 2.74 (1H, dd, J=14 Hz, 10 Hz), 2.99 (1H, dd,J=14 Hz, 4.5 Hz), 3.4-3.8 (4H, m), 4.34-4.50 (1H, m), 4.42 (4H, s),4.66-4.82 (2H, m), 5.42 (4H, brs), 6.50 (1H, s), 6.98 (1H, t, J=6 Hz),7.12-7.28 (5H, m), 7.26 (1H, s), 7.56 (1H, s), 7.80 (1H, s), 7.91 (1H,br), 8.14 (1H, d, J=7.5 Hz), 8.32 (2H, t, J=7.5 Hz), 8.58 (1H, m).

(2) Preparation of7-N-(Glycylglycyl-L-phenylalanylglycyl)aminomethyl-10,11-ethylenedioxy-(20S)-camptothecinHydrochloride

The title compound (164 mg) is obtained in the same manner as in Example1-(9) as a yellow powder from7-N-(t-butoxycarbonylglycylglycyl-L-phenylalanylglycyl)aminomethyl-10,11-ethylenedioxy-(20S)-camptothecin(203 mg).

M.p. >211° C. (decomposed); IR (Nujol): ν_(max) ^(cm−1)=3220, 1745,1655; Mass: m/z=754 ([M−Cl]⁺); NMR (300 MHz, d₆-DMSO): δ^(TMS)=0.88 (3H,t, J=7 Hz), 1.80-1.93 (2H, nm), 2.77 (1H, dd, J=14 Hz, 10 Hz), 3.00 (1H,dd, J=14 Hz, 4 Hz), 3.6-4.55 (7H, m), 4.42 (4H, s), 4.65-4.85 (2H, m),5.42 (2H, s), 5.45 (2H, s), 7.13-7.26 (5H, m), 7.27 (1H, s), 7.57 (1H,s), 7.83 (1H, s), 8.03-8.16 (3H, br), 8.34-8.40 (2H, m), 8.54 (1H, br),8.73 (2H, br).

(3) Preparation of the Camptothecin Derivative

CM-Dextran sodium salt (CM-degree; 0.5) (772 m) is dissolved in water(50 ml), and thereto is added DMF (25 ml). The mixture is stirred underice-cooling, and thereto are added7-N-(glycylglycyl-L-phenylalanylglycyl)amino-methyl-10,11-ethylenedioxy-(20S)-camptothecinhydrochloride (106 mg) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinone(1.57 g). The mixture is reacted overnight, and added into ethanol (450ml) to give the precipitates, which are further treated in the samemanner as in Example 1-(10) to give the desired camptothecin derivative(545 mg) as a pale yellow powder. The content of7-aminomethyl-10,11-ethylenedioxy-(20S)-camptothecin hydrochloride inthe desired camptothecin derivative is 5.5% which is calculated on thebasis of the absorbance at 375 nm. According to the analysis by gelpermeation chromatography (GPC), the average molecular weight of thedesired camptothecin derivative is 165,000, and the degree ofdistribution (Mw/Mn) is 1.40.

Conditions for GPC analysis: G4000SWXL (manufactured by Toso, Ltd), 0.2Mphosphate buffer (pH 7.0).

EXAMPLE 4 Preparation of 10-(3′-Aminopropyloxy)-(20S)-camptothecinHydrochloride

(1) Preparation of5-[3′-(t-Butoxycarbonylamino)propyloxy]-2-nitrobenzaldehyde DimethylAcetal

5-Hydroxy-2-nitrobenzaldehyde dimethyl acetal (3.0 g) is dissolved indry dimethylformamide (50 ml), and thereto are added sodium iodide (3.15g), potassium carbonate (1.93 g) and 3-(t-butoxycarbonylamino)propyltosylate (6.95 g). The mixture is stirred at 50° C. for three hours, andcooled to room temperature. The mixture is extracted with ethyl acetate,and the extract is washed with a saturated aqueous sodium chloridesolution, and dried over sodium sulfate. The resultant is concentratedunder reduced pressure to remove the solvent, and the residue ispurified by silica gel column chromatography to give the title compound(5.22 g) as a pale yellow oil.

Yield: Quantitative; IR (Neat): ν_(max) ^(cm−1)=3360, 1710; Mass:m/z=393 ([M+Na]⁺); NMR (300 MHz, CDCl₃): δ^(TMS)=1.44 (9H, s), 2.02 (2H,quint., J=6 Hz), 3.33 (2H, dd, J=13 Hz, 6 Hz), 3.44 (6H, s), 4.11 (2H,t, J=6 Hz), 4.7 (1H, brs), 6.01 (1H, s), 6.90 (1H, dd, J=9 Hz, 3 Hz),7.29 (1H, d, J=3 Hz), 7.97 (1H, d, J=9 Hz).

(2) Preparation of10-[3′-(t-Butoxycarbonylamino)propyloxy]-(20S)-camptothecin

5-[3′-(t-Butoxycarbonylamino)propyloxy]-2-nitrobenzaldehyde dimethylacetal (1270 mg) is dissolved in ethanol (20 ml), and thereto is added10% palladium-carbon (120 mg), and the mixture is stirred under hydrogenatmosphere for 1.5 hour. The catalyst is removed by filtration, and tothe filtrate are added(4S)-7,8-dihydro-4-ethyl-4-hydroxy-1H-pyrano[3,4-f]-indolidine-3,6,10(4H)-trione(300 mg) and p-toluenesulfonic acid (22 mg), and the mixture is stirredat room temperature overnight. The mixture is concentrated under reducedpressure to remove the solvent, and the residue is purified by silicagel column chromatography to give the title compound (204 mg) as a paleyellow powder.

M.p. 223-224° C. (decomposed); Yield: 34%; IR (Nujol): ν_(max) ^(cm)⁻¹=3360, 1750, 1690; Mass: m/z=522 ([M+H]⁺); NMR (300 MHz, CDCl₃):δ^(TMS)=1.03 (3H, t, J=7.5 Hz), 1.46 (9H, s), 1.8-2.0 (2H, m), 2.08 (1H,dd, J=12.5 Hz, 6.5 Hz), 2.10 (1H, dd, J=12.5 Hz, 6 Hz), 3.40 (2H, q,J=6.5 Hz), 4.18 (2H, t, J=6 Hz), 4.82 (1H, brs), 5.24 (2H, s), 5.29 (1H,d, J=16 Hz), 5.73 (1H, d, J=16 Hz), 7.12 (1H, d, J=3 Hz), 7.43 (1H, dd,J=9 Hz, 3 Hz), 7.61 (1H, s), 8.09 (1H, d, J=9 Hz), 8.20 (1H, d, J=9Hz).

(3) Preparation of 10-(3′-Aminopropyloxy)-(20S)-camptothecinHydrochloride

10-[3′-(t-Butoxycarbonylamino)propyloxy]-(20S)-camptothecin (352 mg) isdissolved in dry dioxane-ethanol (7 ml-1 ml), and to the mixture isadded with stirring 19% hydrochloric acid in dioxane (5 ml) underice-cooling. The reaction mixture is stirred at room temperature, andthereto is added isopropyl ether (10 ml). The precipitated powder iscollected by filtration, and washed to give the title compound (339 mg)as a yellow powder.

M.p. 214-218° C. (decomposed); IR (Nujol): ν_(max) ^(cm−1)=3470, 3280,1745; Mass: m/z=422 ([M−Cl]⁺); NMR (300 MHz, d₆-DMSO): δ^(TMS)=0.89 (3H,t, J=7.5 Hz), 1.80-1.95 (2H, m), 2.10-2.22 (2H, m), 2.96-3.10 (2H, m),4.27 (2H, t, J=6 Hz), 5.25 (2H, s), 5.42 (2H, s), 7.29 (1H, s),7.49-7.55 (2H, m), 8.08 (1H, d, J=10 Hz), 8.19 (3H, brs), 8.54 (1H, s).

EXAMPLE 5 Preparation of10-[3′-(Glycyl-glycyl-L-phenylalanyl-glycylamino)propyloxy]-(20S)-camptothecinHydrochloride

(1) Preparation of10-[3′-(t-Butoxycarbonyl-glycyl-glycyl-L-phenylalanyl-glycylamino)propyloxy]-(20S)-camptothecin

10-(3′-Aminopropyloxy)-(20S)-camptothecin hydrochloride (325 mg) isdissolved in dry dimethylformamide (10 ml), and thereto are added withstirring N-hydroxysuccinimide (4 equivalents), diisopropylethylamine (2equivalents), N-t-butoxycarbonyl-glycyl-glycyl-L-phenylalanylglycine (2equivalents) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (4 equivalents) under ice-cooling. The reaction mixture isstirred at room temperature overnight, and concentrated under reducedpressure to remove the solvent. The residue is purified by silica gelcolumn chromatography to quantitatively give the title compound (597 mg)as a yellow powder.

IR (Nujol): ν_(max) ^(cm−1)=3280, 1750, 1660; Mass: m/z=840 ([M+H]⁺);NMR (300 MHz, d₆-DMSO): δ^(TMS)=0.88 (3H, t, J=7 Hz), 1.36 (9H, s),1.8-2.04 (4H, m), 2.72-2.84 (1H, m), 3.00-3.12 (1H, m), 3.24-3.36 (2H,m), 3.50-3.80 (6H, m), 4.18 (2H, t, J=6 Hz), 4.44-4.54 (1H, m), 5.24(2H, s), 5.42 (2H, s), 6.50 (1H, s), 6.99 (1H, t, J=6 Hz), 7.12-7.27(5H, m), 7.28 (1H, s), 7.48-7.55 (1H, m), 7.50 (1H, s), 7.88-7.96 (1H,m), 8.07 (1H, d, J=9 Hz), 8.12-8.36 (2H, m), 8.51 (1H, s).

(2) Preparation of10-[3′-(Glycyl-glycyl-L-phenylalanyl-glycylamino)propyl-oxy]-(20S)-camptothecinHydrochloride

10-[3′-(t-Butoxycarbonyl-glycyl-glycyl-L-phenylalanyl-glycylamino)-propyloxy]-(20S)-camptothecin(580 mg) is treated in the same manner as in Example 4-(3) to give thetitle compound (438 mg) as a yellow powder.

Yield: 82%; M.p. 194-199° C. (decomposed); IR (Nujol): ν_(max)^(cm−1)=3190, 1745, 1650; Mass: m/z=740 ([M−Cl]⁺); NMR (300 MHz,d₆-DMSO): δ^(TMS)=0.88 (3H, t, J=7 Hz), 1.80-2.03 (4H, m), 2.79 (1H, dd,J=14 Hz, 10 Hz), 3.05 (1H, dd, J=14 Hz, 5 Hz), 3.2-3.5 (2H, m),3.52-3.62 (2H, m), 3.62-3.83 (4H, m), 4.19 (2H, t, J=6 Hz), 4.48-4.58(1H, m), 5.25 (2H, s), 5.42 (2H, s), 6.5 (1H, brs), 7.13-7.26 (5H, m),7.28 (1H, s), 7.49-7.55 (1H, m), 7.50 (1H, s, J=9.5 Hz), 7.93 (1H, t,J=6 Hz), 8.0-8.14 (4H, m), 8.32-8.41 (2H, m), 8.51 (1H, s), 8.56 (1H, t,J=5.5 Hz).

EXAMPLE 6 Preparation of the Camptothecin Derivative of the FollowingFormula (Where -Gly-Gly-L-Phe-Gly- is SEQ ID NO: 3)

CM-Dextran sodium salt (CM-degree; 0.5) (513 mg) is dissolved in water(50 ml), and thereto are added with stirring10-[3′-(glycyl-glycyl-L-phenylalanyl-glycylamino)propyloxy]-(20S)-camptothecinhydrochloride (77 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (1.54 g), during which the butch temperature is kept at atemperature below 10° C. The mixture is stirred for two hours at a butchtemperature of below 10° C., during which the pH value of the mixture iskept at pH 6.0-6.5 with 0.2N hydrochloric acid. The reaction mixture ispurified by ion-exchange column chromatography (AGMP-50, Na-type, 30 ml,manufactured by Bio-Rad, Laboratories, Inc.). The fractions containingthe desired compound are combined, filtered, and to the filtrate isadded ethanol. The precipitates are collected by centrifugation, washed,and dried under reduced pressure to give a pale yellow powdery complex(492 mg). The content of 10-(3′-aminopropyloxy)-(20S)-camptothecinhydrochloride in the desired camptothecin derivative is 2.8% which iscalculated on the basis of the absorbance at 380 nm. According to theanalysis by gel permeation chromatography (GPC), the average molecularweight of the desired camptothecin derivative is 179,000, and the degreeof distribution (Mw/Mn) is 1.42.

Conditions for GPC analysis: G4000SWXL (manufactured by Toso,

Ltd), 0.2M phosphate buffer (pH 7.0).

EXAMPLE 7 Preparation of the Camptothecin Derivative of the FollowingFormula (Where -Gly-Gly-L-Phe-Gly- is SEQ ID NO: 3)

(1) Preparation of(9S)-1-(t-Butoxycarbonylglycylglyclyl-L-phenylalanyl-glycylamino)-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo-[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13[9H,15H]-dione

The title compound (247 mg) is obtained in the same manner as in Example1-(8) as a pale yellow amorphous solid from(9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]-indolidino[1,2-b]quinoline-10,13[9H,15H]-dionehydrochloride (166 mg) andt-butoxycarbonylglycylglyclyl-L-phenylalanylglycine (2 equivalents).

Yield: 82%; IR (Nujol): ν_(max) ^(cm−1)=3290, 1710, 1655; Mass: m/z=854([M+H]⁺); NMR (300 MHz, d₆-DMSO): δ^(TMS)=0.87 (3H, t, J=7 Hz), 1.37(9H, s), 1.8-1.95 (2H, m), 2.05-2.3 (1H, m), 2.42 (3H, s), 2.5-2.85 (2H,m), 2.9-3.1 (1H, m), 3.15-3.4 (2H, m), 3.5-3.8 (6H, m), 4.4-4.55 (1H,m), 5.26 (2H, s), 5.42 (2H, s), 5.55-5.65 (1H, m), 6.53 (1H, s), 6.99(1H, t, J=5 Hz), 7.1-7.3 (5H, m), 7.32 (1H, s), 7.81 (1H, d, J=11 Hz),7.8-7.95 (1H, m), 8.1-8.2 (1H, m), 8.3-8.4 (1H, m), 8.4-8.5 (1H, m).

(2) Preparation of(9S)-1-(Glycyl-glycyl-L-phenylalanylglycylamino)-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano-[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13[9H,15H]-dione

The title compound (193 mg) is obtained in the same manner as in Example1-(9) as a yellow powder from the compound (220 mg) in the above (1).

M.p. >165+ C. (decomposed); IR (Nujol): ν_(max) ^(cm−1)=3350, 1745,1660, 1615; Mass: m/z=754 ([M−Cl+H]⁺); NMR (300 MHz, d₆-DMSO):δ^(TMS)=0.87 (3H, t, J=7 Hz), 1.80-1.94 (2H, m), 2.08-2.27 (2H, m), 2.41(3H, s), 2.77 (1H, dd, J=13 Hz, 9 Hz), 3.01 (1H, dd, J=13 Hz, 5 Hz),3.15-3.28 (2H, m), 3.5-3.91 (6H, m), 4.45-4.56 (1H, m), 5.25 (2H, s),5.41 (1H, d, J=13 Hz), 5.42 (1H, d, J=13 Hz), 5.57 (1H, m), 7.12-7.30(5H, m), 7.32 (1H, s), 7.80 (1H, d, J=11 Hz), 8.0-8.2 (3H, br), 8.32(1H, d, J=7 Hz), 8.43 (1H, t, J=5.5 Hz), 8.50-8.62 (2H, m).

(3) Preparation of the Camptothecin Derivative

CM-Dextran sodium salt (CM-degree; 0.65) (2000 mg) and the compound (170mg) obtained in the above (2) are treated in the same manner as inExample 3-(3) to give the desired camptothecin derivative (1803 mg) as apale yellow powder. The content of(9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13[9H,15H]-dionehydrochloride in the desired camptothecin derivative is 3.0% which iscalculated on the basis of the absorbance at 376 nm. According to theanalysis by gel permeation chromatography (GPC), the average molecularweight of the desired camptothecin derivative is 187,000, and the degreeof distribution (Mw/Mn) is 1.54.

Conditions for GPC analysis: G4000SWXL (manufactured by Toso, Ltd), 0.2Mphosphate buffer (pH 7.0).

EXAMPLES 8-24

The camptothecin derivatives as listed in Table 1 are obtained in thesame manner as in Examples 1-6.

TABLE 1

Ex. No. R 8 CM.Dextran.Na—L-Phe- 9 CM.Dextran.Na—Gly-Gly- 10CM.Dextran.Na—L-Leu-Gly- 11 CM.Dextran.Na—L-Phe-Gly- 12CM.Dextran.Na—L-Tyr-Gly- 13 CM.Dextran.Na—Gly-Gly- Gly- 14CM.Dextran.Na—Gly-Gly- Gly-Gly- 15 CM.Dextran.Na—Gly-Gly- Gly-Gly-Gly-16 CM.Pullulan.Na—L-Phe- 17 CM.Pullulan.Na—Gly-Gly- 18CM.Pullulan.Na—L-Leu-Gly- 19 CM.Pullulan.Na—L-Phe-Gly- 20CM.Pullulan.Na—L-Tyr-Gly- 21 CM.Pullulan.Na—Gly-Gly- L-Phe-Gly- 22CM.Pullulan.Na—Gly-Gly- Gly- 23 CM.Pullulan.Na—Gly-Gly- Gly-Gly- 24CM.Pullulan.Na—Gly-Gly- Gly-Gly-Gly- [CM.Pullulan.Na:Carboxymethylpullulan sodium salt] Where: -Gly-Gly-Gly-Gly- is SEQ IDNO:1; -Gly-Gly-Gly-Gly-Gly- is SEQ ID NO:2; and -Gly-Gly-L-Phe-Gly- isSEQ ID NO:3

EXAMPLES 25-41

The camptothecin derivatives as listed in Table 2 are obtained in thesame manner as in Examples 1-6.

TABLE 2

Ex. No. R 25 CM.Dextran.Na—L-Phe- 26 CM.Dextran.Na—Gly-Gly- 27CM.Dextran.Na—L-Leu-Gly- 28 CM.Dextran.Na—L-Phe-Gly- 29CM.Dextran.Na—L-Tyr-Gly- 30 CM.Dextran.Na—Gly-Gly- Gly- 31CM.Dextran.Na—Gly-Gly- Gly-Gly- 32 CM.Dextran.Na—Gly-Gly- Gly-Gly-Gly-33 CM.Pullulan.Na—L-Phe- 34 CM.Pullulan.Na—Gly-Gly- 35CM.Pullulan.Na—L-Leu-Gly- 36 CM.Pullulan.Na—L-Phe-Gly- 37CM.Pullulan.Na—L-Tyr-Gly- 38 CM.Pullulan.Na—Gly-Gly- L-Phe-Gly- 39CM.Pullulan.Na—Gly-Gly- Gly- 40 CM.Pullulan.Na—Gly-Gly- Gly-Gly- 41CM.Pullulan.Na—Gly-Gly- Gly-Gly-Gly- Where: -Gly-Gly-Gly-Gly- is SEQ IDNO:1; -Gly-Gly-Gly-Gly-Gly- is SEQ ID NO:2; and -Gly-Gly-L-Phe-Gly- isSEQ ID NO:3

EXAMPLES 42-59

The camptothecin derivatives as listed in Table 3 are obtained in thesame manner as in Examples 1-6.

TABLE 3

Ex. No. R 42 CM.Dextran.Na—L-Phe- 43 CM.Dextran.Na—Gly-Gly- 44CM.Dextran.Na—L-Leu-Gly- 45 CM.Dextran.Na—L-Phe-Gly- 46CM.Dextran.Na—L-Tyr-Gly- 47 CM.Dextran.Na—Gly-Gly- L-Phe-Gly- 48CM.Dextran.Na—Gly-Gly-Gly- 49 CM.Dextran.Na—Gly-Gly- Gly-Gly- 50CM.Dextran.Na—Gly-Gly- Gly-Gly-Gly- 51 CM.Pullulan.Na—L-Phe- 52CM.Pullulan.Na—Gly-Gly- 53 CM.Pullulan.Na—L-Leu-Gly- 54CM.Pullulan.Na—L-Phe-Gly- 55 CM.Pullulan.Na—L-Tyr-Gly- 56CM.Pullulan.Na—Gly-Gly- L-Phe-Gly- 57 CM.Pullulan.Na—Gly-Gly- Gly- 58CM.Pullulan.Na—Gly-Gly- Gly-Gly- 59 CM.Pullulan.Na—Gly-Gly- Gly-Gly-Gly-Where: -Gly-Gly-Gly-Gly- is SEQ ID NO:1; -Gly-Gly-Gly-Gly-Gly- is SEQ IDNO:2; and -Gly-Gly-L-Phe-Gly- is SEQ ID NO:3

EXAMPLES 60-76

The camptothecin derivatives as listed in Table 4 are obtained in thesame manner as in Examples 1-6.

TABLE 4

Ex. No. R 60 CM.Dextran.Na—L-Phe- 61 CM.Dextran.Na—Gly-Gly- 62CM.Dextran.Na—L-Leu-Gly- 63 CM.Dextran.Na—L-Phe-Gly- 64CM.Dextran.Na—L-Tyr-Gly- 65 CM.Dextran.Na—Gly-Gly-Gly- 66CM.Dextran.Na—Gly-Gly- Gly-Gly- 67 CM.Dextran.Na—Gly-Gly- Gly-Gly-Gly-68 CM.Pullulan.Na—L-Phe- 69 CM.Pullulan.Na—Gly-Gly- 70CM.Pullulan.Na—L-Leu-Gly- 71 CM.Pullulan.Na—L-Phe-Gly- 72CM.Pullulan.Na—L-Tyr-Gly- 73 CM.Pullulan.Na—Gly-Gly- L-Phe-Gly- 74CM.Pullulan.Na—Gly-Gly- Gly- 75 CM.Pullulan.Na—Gly-Gly- Gly-Gly- 76CM.Pullulan.Na—Gly-Gly- Gly-Gly-Gly- Where: -Gly-Gly-Gly-Gly- is SEQ IDNO:1; -Gly-Gly-Gly-Gly-Gly- is SEQ ID NO:2; and -Gly-Gly-L-Phe-Gly- isSEQ ID NO:3

EXAMPLES 77-93

The camptothecin derivatives as listed in Table 5 are obtained in thesame manner as in Examples 1-6.

TABLE 5

Ex. No. R 77 CM.Dextran.Na—L-Phe- 78 CM.Dextran.Na—Gly-Gly- 79CM.Dextran.Na—L-Leu-Gly- 80 CM.Dextran.Na—L-Phe-Gly- 81CM.Dextran.Na—L-Tyr-Gly- 82 CM.Dextran.Na—Gly-Gly- Gly- 83CM.Dextran.Na—Gly-Gly- Gly-Gly- 84 CM.Dextran.Na—Gly-Gly- Gly-Gly-Gly-85 CM.Pullulan.Na—L-Phe- 86 CM.Pullulan.Na—Gly-Gly- 87CM.Pullulan.Na—L-Leu-Gly- 88 CM.Pullulan.Na—L-Phe-Gly 89CM.Pullulan.Na—L-Tyr-Gly- 90 CM.Pullulan.Na—Gly-Gly- L-Phe-Gly- 91CM.Pullulan.Na—Gly-Gly- Gly- 92 CM.Pullulan.Na—Gly-Gly- Gly-Gly- 93CM.Pullulan.Na—Gly-Gly- Gly-Gly-Gly- Where: -Gly-Gly-Gly-Gly- is SEQ IDNO:1; -Gly-Gly-Gly-Gly-Gly- is SEQ ID NO:2; and -Gly-Gly-L-Phe-Gly- isSEQ ID NO:3

EXAMPLES 94-110

The camptothecin derivatives as listed in Table 6 are obtained in thesame manner as in Examples 1-6.

TABLE 6

Ex. No. R 94 CM.Dextran.Na—L-Phe- 95 CM.Dextran.Na—Gly-Gly- 96CM.Dextran.Na—L-Leu-Gly- 97 CM.Dextran.Na—L-Phe-Gly- 98CM.Dextran.Na—L-Tyr-Gly- 99 CM.Dextran.Na—Gly-Gly- Gly- 100CM.Dextran.Na—Gly-Gly- Gly-Gly- 101 CM.Dextran.Na—Gly-Gly- Gly-Gly-Gly-102 CM.Pullulan.Na—L-Phe- 103 CM.Pullulan.Na—Gly-Gly- 104CM.Pullulan.Na—L-Leu-Gly- 105 CM.Pullulan.Na—L-Phe-Gly 106CM.Pullulan.Na—L-Tyr-Gly- 107 CM.Pullulan.Na—Gly-Gly- L-Phe-Gly- 108CM.Pullulan.Na—Gly-Gly- Gly- 109 CM.Pullulan.Na—Gly-Gly- Gly-Gly- 110CM.Pullulan.Na—Gly-Gly- Gly-Gly-Gly- Where: -Gly-Gly-Gly-Gly- is SEQ IDNO:1; -Gly-Gly-Gly-Gly-Gly- is SEQ ID NO:2; and -Gly-Gly-L-Phe-Gly- isSEQ ID NO:3

REFERENCE EXAMPLE 1

(1) Dextran (Dextran T-110, average molecular weight; 100,000 (by theGPC analysis), manufactured by Pharmacia Biotech AB) (29 g) is dissolvedin water (290 ml). To the solution is added sodium borohydride (1.45 g)at 0-5° C., and the mixture is stirred at 5+ C. overnight. The pH valueof the reaction mixture is adjusted to pH 5 with acetic acid, and themixture is further stirred at room temperature for 3 hours. The pH valueof the mixture is adjusted to pH 7 with 2N aqueous sodium hydroxidesolution, and thereto is added ethanol (1.2 L) with vigorously stirring.The mixture is allowed to stand, and the insoluble materials areprecipitated. The supernatant of the mixture is removed by decantation,and the residue is centrifuged. The residue is dissolved in water (0.5L) and the mixture is lyophilized to give a white powder (26.3 g).

(2) The white powder thus obtained (50 g) is dissolved in water (500ml), and thereto is added sodium hydroxide (200 g) under ice-cooling.The mixture is stirred for 30 minutes, and warmed to room temperature.To the mixture is added dropwise a solution of monochloroacetic acid(110 g) in water (150 ml), and the mixture is stirred at 40° C. for 18hours. The reaction mixture is cooled to a temperature below 10° C., andthe pH value of the mixture is adjusted to pH 8-9 with acetic acid.Methanol (8 L) is added to the reaction mixture with vigorouslystirring, and the insoluble materials are precipitated. The insolublematerials are collected by filtration, and dissolved in pure water (5L). The solution is desalted by ultrafiltration. The residual solutionis concentrated under reduced pressure, and filtered. Ethanol is addedto the filtrate, and the precipitated material is collected byfiltration, washed with aqueous ethanol and acetone, and dried underreduced pressure at room temperature, and then dried under reducedpressure at 50° C. to give carboxymethyldextran (CM-dextran) sodium salt(the degree of carboxymethylation by neutralization titration method;0.5) (50.2 g).

REFERENCE EXAMPLE 2

CM-Dextran sodium salt having a degree of carboxymethylation of 0.65 isobtained in the same manner as in Reference Example 1 except that theamount of monochloroacetic acid is changed.

4 1 4 PRT Artificial Sequence Peptide which intervenes between acamptothecin compound [I] and a polysaccharide having carboxyl groups. 1Gly Gly Gly Gly 1 2 5 PRT Artificial Sequence Peptide which intervenesbetween a camptothecin compound [I] and a polysaccharide having carboxylgroups. 2 Gly Gly Gly Gly Gly 1 5 3 4 PRT Artificial Sequence Peptidewhich intervenes between a camptothecin compound [I] and apolysaccharide having carboxyl groups. 3 Gly Gly Phe Gly 1 4 4 PRTArtificial Sequence Peptide which intervenes between a camptothecincompound [I] and a polysaccharide having carboxyl groups. 4 Gly Gly PheGly 1

What is claimed is:
 1. A compound comprising a camptothecin compoundrepresented by the formula (I):

wherein any two substituent variables R¹, R², R³, R⁴ and R⁵ that areadjacent to one another combine to form an alkylene group, two of theremaining three substituent variables among R¹, R², R³, R⁴ and R⁵ arehydrogen, and the remaining substituent variable among R¹, R², R³, R⁴and R⁵ is a group of the formula: —X_(n)-Alk_(m)-R⁶, wherein one or twomethylene groups of the alkylene group is optionally replaced with —O—,—S— or —NH—, X is —O— or —NH—, Alk is an alkylene group, R⁶ is —NH—R⁷, agroup of the formula:

m and n are both 0 or 1, or m is 1 and n is 0, and R⁷ is an amino acidor peptide, provided that R³ and R⁴ do not combine to form amethylenedioxy group; or a salt thereof.
 2. The compound according toclaim 1, wherein R⁷ is bonded via an amide or ester bond; or a saltthereof.
 3. The compound according to claim 2, wherein the C-terminalcarboxyl group of the amino acid or the peptide of R⁷ is bonded throughan amide or ester bond; or a salt thereof.
 4. The compound according toclaim 3, wherein R⁶ of the compound (I) is —NH—R⁷ or a group of theformula:

wherein R⁷ is a peptide; or a salt thereof.
 5. The compound according toclaim 4, wherein (1) R¹ and R² combine to form a trimethylene group, R³is a 3-aminopropyloxy group, R⁴ and R⁵ are each a hydrogen atom; (2) R¹is a piperazinomethyl group, R² and R⁵ are each a hydrogen atom, R³ andR⁴ combine to form an ethylenedioxy group; (3) R¹ is an aminomethylgroup, R² and R⁵ are each a hydrogen atom, R³ and R⁴ combine to form anethylenedioxy group; or a salt thereof.
 6. The compound according toclaim 5, wherein the peptide is selected from the group consisting ofglycyl-glycyl-L-phenylalanyl-glycine (SEQ ID NO:3),glycyl-glycyl-D-phenylalanyl-glycine (SEQ ID NO:4), glycyl-glycine,glycyl-glycyl-glycine, glycyl-glycyl-glycyl-glycine (SEQ ID NO:1),glycyl-glycyl-glycyl-glycyl-glycine (SEQ ID NO:2), L- orD-phenylalanyl-glycine and L- or D-leucyl-glycine; or a salt thereof. 7.The compound according to claim 6, wherein the peptide isglycyl-glycyl-L-phenylalanyl-glycine (SEQ ID NO:3); or a salt thereof.8. The compound according to claim 6, wherein the peptide isglycyl-glycine; or a salt thereof.
 9. The compound according to claim 6,wherein the peptide is glycyl-glycyl-glycine; or a salt thereof.
 10. Thecompound according to claim 6, wherein the peptide isglycyl-glycyl-glycyl-glycine (SEQ ID NO:1); or a salt thereof.
 11. Thecompound according to claim 6, wherein the peptide is L- orD-phenylalanyl-glycine; or a salt thereof.
 12. The compound according toclaim 1, wherein said compound is as follows: